Chimeric enzyme for host nucleic acid autohydrolysis

描述（由申请人提供）： DNA疫苗和基因药物正在成为一类重要的新型药物，它们来源于细菌质粒。为了用于人类，质粒DNA应基本上不含细菌基因组DNA和RNA、蛋白质和内毒素。假设E.大肠杆菌质粒DNA可以通过在质粒产生菌株中共表达RNase和DNase而更有效地纯化。该策略将是制造杂交蛋白（表达RNase A和噬菌体T5 DNA外切核酸酶）并将它们分泌到周质空间中，在适当的时间释放它们以允许E. coli RNA和基因组DNA。在特定目标I中，我们将构建用于表达RNase和T5核酸外切酶的重组基因。在具体目标II中，我们将研究重组酶在质粒纯化中的应用。拟议的I期可行性研究的总体目标是确定这种细菌菌株在质粒DNA纯化中是否具有显著优势。如果成功，II期研究将包括将基因插入E.大肠杆菌染色体;创造所需的菌株;开发第三种酶（蛋白酶）;以及开发一种诱导系统，用于在发酵的最后几个小时将外源蛋白引入细胞质。在第三阶段，NTC将在商业质粒生产中引入改进的菌株。 成功构建一种或多种以可控方式表达RNA酶、DNA酶和/或蛋白酶的有用细菌菌株（里程碑II）将是商业规模DNA纯化的重大发展。这些菌株将被迅速引入NTC的大规模DNA制造过程，并通过该公司的技术转让和许可计划向其他商业和学术用户提供许可。目前，实验性基因药物和DNA疫苗通常以克为单位生产，主要用于动物安全性和有效性研究。然而，FDA批准的商业产品的出现将需要扩大到千克，例如，对于广泛使用的疫苗。从目前每克DNA生产22，000 - 46，000美元的价格来看，迫切需要一种成本更低、可扩展的发酵工艺。由此产生的产品的经济效益和潜在市场规模是显而易见的。使用内源产生的重组酶的能力将是优于使用动物来源的或外源添加的酶的显著优势（在安全性和经济性方面）。从拟议的工作中产生商业上有用和有价值的产品的可能性被认为很高。

DESCRIPTION (provided by applicant): DNA vaccines and gene medicines are emerging as an important new class of pharmaceuticals that are derived from bacterial plasmids. For use in humans, plasmid DNA should be essentially free from bacterial genomic DNA and RNA, protein and endotoxin. The hypothesis is that E. coli plasmid DNA can be more efficiently purified by co-expressing RNase and DNase in plasmid producing strains of bacteria. The strategy will be to make hybrid proteins (expressing RNase A and phage T5 DNA exonuclease) and secrete them into the periplasmic space, releasing them at the appropriate time to allow digestion of E. coli RNA and genomic DNA. In Specific Aim I, we will construct recombinant genes for expression of RNase and T5 exonuclease. In Specific Aim II, we will investigate the use of the recombinant enzymes in the purification of plasmids. The overall goal of the proposed Phase I feasibility study is to determine whether such a bacterial strain is a significant advantage in the purification of plasmid DNA. If successful, Phase II studies will include improved constructs for insertion of the genes into the E. coli chromosome; creating the strains needed; development of a third enzyme (protease); and development of an inducible system for introducing the foreign proteins into the cytoplasm during the final hours of fermentation. In Phase Ill, NTC will introduce the improved strains in commercial plasmid production. Success in constructing one or more useful strains of bacteria expressing RNAse, DNAse and/or protease enzymes in a controllable fashion (milestone II) would be a significant development for DNA purification on a commercial scale. Such strains will be rapidly introduced into NTC's large scale DNA manufacturing process, and will also be made available for licensing to other commercial and academic users through the company's technology transfer and licensing program. Currently, experimental gene drugs and DNA vaccines are routinely made on the gram scale, mostly for use in animal safety and efficacy studies. However, the advent of FDA approved commercial products will require scaling up to kilograms, for example, for a widely used vaccine. Judging from the current price of $22,000- $46,000/gram of DNA produced, a less costly, scalable fermentation process is badly needed. The economic benefits and potential market size for the resulting products can be readily appreciated. The ability to use endogenously produced recombinant enzymes would be a significant advantage (in terms of safety and economy) over the use of animal-derived or exogenously added enzymes. The likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.